We have examined the DNA sequences responsible for regulation of insulin expression in the Beta-cells of pancreas. A DNA transfection procedure for introducing DNA molecules into established lines of transformed hamster Beta-cells (HIT) has been established in the laboratory. Using this protocol, a sequence upstream of the rat insulin I gene has been identified which can activate expression from a heterologous promoter to high levels in the HIT line. This element shares several properties with viral enhancer elements and is presently being characterized in detail. Current studies also focus on the identification of other transcriptional regulatory elements, such as promoter sequences responsible for tissue-specific expression of insulin. In addition, upstream sequences may contain signals inhibitory for transcription. Ongoing studies will further map and characterize the potential inhibitory region and investigate their mode of action.